REVISED ABSTRACT Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder marked by inattention, hyperactivity and impulsivity. Stimulant therapies can be effective for alleviating ADHD symptoms; however, due to neurobiological heterogeneity of ADHD, some patients are non-responders, or develop undesirable side effects. This creates a process of trial-and-error in the clinical pursuit of alternative treatment options. Currently there are no widely utilized laboratory tests that measure clinically actionable neuroendocrine analytes in ADHD that can provide adequate prediction of treatment success and remission. This project will develop a companion diagnostic for ADHD to complement existing symptom evaluation questionnaires, which are indirect measures of treatment outcome. The proposed companion diagnostic encompasses developing a clinical test for neuroendocrine molecules ? GABA, allopregnanolone, glutamate, serotonin and kynurenine analyzed in dried blood spot (DBS) due to its minimally-invasive nature. We will use pre-existing whole blood samples from children with ADHD collected as part of an active randomized control trial by a team of academic collaborators at Oregon Health & Science University to evaluate and confirm dysregulated neuroendocrine tone in ADHD. Specific aims: 1. A) Develop and validate an analytical assay for GABA and allopregnanolone in DBS. We will determine analytical parameters, including linearity, recovery, precision, etc., and confirm accuracy by comparing DBS to liquid blood analysis, which is the currently accepted gold standard method. 1. B) Incorporate glutamate, serotonin and kynurenine analytes for simultaneous analysis. 2. Evaluate the performance of the clinical test using DBS samples from medication-free pediatric patients with ADHD. Completion of these aims will serve as proof-of-concept for a fully validated DBS test for baseline (no treatment) levels of GABA, allopregnanolone, glutamate, serotonin and kynurenine in ADHD. This will establish the basis for Phase II, developing a comprehensive clinical study that will examine the changes in neuroendocrine parameters in response to stimulants, non-stimulants and nutritional supplements used in ADHD.